Microfilaments are found In essentially all eucaryotic cells where they function to provide structural, yet dynamic, Support for basic cellular processes. We have initiated a project to identify the molecular components of the microfilament-based cytoskeleton in Saccharomyces cerevisiae and to use the excellent molecular genetics available in this organism to probe the functions of each component. We have found that yeast contains a single gene encoding a protein with many of the characteristic properties of higher cell tropomyosins. Disruption of the tropomyosin gene, designated TPM1, in yeast is not lethal but confers slower growth, an aberrant actin cytoskeleton, and the accumulation of abundant Intracellular vesicles. We propose to use genetic and biochemical approaches to identify proteins that function together with tropomyosin in yeast. Genetic screens will identify mutations which become lethal only in cells depleted of tropomyosin. These synthetic lethal mutations will identify a set of genes that will be cloned, sequenced and the physiological consequences of their disruption analyzed. We shall also generate mutations in the TPM1 gene that make a lethal protein, poisoning processes in which tropomyosin is involved. This shall allow a direct identification of these processes. Proteins that interact physically with tropomyosin will be identified biochemically, purified and characterized. Genes specifying these proteins will be cloned and sequenced and subjected to genetic analysis. The vesicles that accumulate in tropomyosin-deficient cells will be purified and characterized to determine in which intracellular vesicular pathway they lie. It is also proposed to continue two other projects. We are interested in studying proteins that modulate the ration of actin monomer to polymer. We will extend our analysis of the AMB1 gene, whose product binds monomeric actin. We will also make use of the fact that over-expression of the single actin gene, ACT1, is lethal. Over-expression of proteins that bind monomeric actin may be able to suppress this lethality. We have constructed and characterized a yeast regulated cDNA expression library; we shall use this library to identify yeast cDNA clones whose over-expression can suppress the lethality associated with actin over-expression. We shall also use this library to identify proteins whose over-expression interferes with the normal function of the actin cytoskeleton. These studies should greatly advance our goal of uncovering the function of actin and its associated proteins in yeast.